Numerous regulatory genes and environmental cues that influence Streptococcus pyogenes virulence gene regulation in vitro have been identified. However, very little is currently known as to how these signals are sensed, how different regulatory pathways interact and whether these cues are relevant in vivo. These are important questions, as differential regulation in response to compartment-specific cues may determine whether infection proceeds to a self-limiting or tissue-destructive outcome, an issue that is only very poorly understood for S. pyogenes. To gain insight into these questions, my lab has focused on regulation of speB, which encodes the secreted SpeB cysteine protease. We defined a set of in vitro conditions, including growth phase, pH, Cl- anion concentration and a carbohydrate-poor/peptide rich nutritional environment, that reflects expression patterns measured in vivo. Mutagenesis to identify a regulatory factor that could coordinate speB regulation in response to each of these cues resulted in the discovery of LacD.1, a tagatose aldolase that acts to repress speB transcription. Interestingly, S. pyogenes and several other Gram-positive pathogens contain two lactose operons (Lac.1 and Lac.2) where several of the genes encoding enzymes upstream of LacD.1, but not LacD.2, in the catabolic pathway are missing or are pseudogenes, suggesting that Lac.2 is involved in catabolism and that Lac.1 has evolved to a regulatory function. Consistent with this, LacD.2 has no regulatory phenotype and cannot complement the regulatory phenotype of LacD.1 and mutations that disrupt the catalytic center of LacD.1 do not alter its regulatory function;however, other mutations that may alter its ability to bind substrate do ablate regulation. Furthermore, we have shown that LacD.1 forms a complex in vivo with RopB, a DMA-binding protein and a known regulator of transcription and other metabolic genes. RopB is a member of the Rgg-family of transcription regulators broadly distributed among Gram-positive pathogens and virtually nothing is known about how this important family of regulators interacts with signal transduction systems. Based on examples of how other aldolases and sugar catabolic enzymes have been adapted to regulatory functions, these data suggest the following model for LacD.1 function: 1. That LacD.1 has been adapted as a sensor of intermediary metabolism;2. That under carbohydrate-rich conditions, binding its substrate allows LacD.1 to act as an "anti-activator" and sequester RopB in an inactive form;3. That LacD.1 may play a broader role in carbon catabolite repression and virulence gene expression;and 4. that LacD.1 is important for virulence. This project will explore these questions